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MAC1 AS A POTENTIAL MARKER FOR FUNCTIONAL HEMATOPOIETIC STEM CELLS DURING EARLY REGENERATION AFTER TRANSPLANTATION
S84
Poster Presentations / Experimental Hematology 2019;76 (Suppl):S52−S96
3118 - MAC1 AS A POTENTIAL MARKER FOR
FUNCTIONAL HEMATOPOIETIC STEM CELLS DURING EARLY REGENERATION AFTER TRANSPLANTATION Anna Rydstrom, Jonas Larsson Lund University, Lund, Sweden
Mouse hematopoietic stem cells (HSCs) can be isolated to high purity using Lineage-, Sca1+, cKit+, CD48-, CD150+ (LSK SLAM) as markers. Cycling HSCs in the fetal liver have been shown to express the CD11b/CD18 Macrophage-1 antigen (Mac1), a marker expressed mostly on mature cells in the adult bone marrow (Weissman et al., 1995). Furthermore, Mac1 is upregulated in activated adult HSCs after treatment with the cytotoxic drug 5-fluorouracil. For this reason, Mac1 should be omitted from the lineage staining when analyzing fetal liver and 5-FU treated mice. Here we asked whether Mac1 expression is altered also during reconstitution after transplantation into irradiated recipients, and if it can be used to further define the functional HSC compartment in a state of active regeneration. To address this question, LSK cells were transplanted into lethally irradiated (900cGy) primary recipient mice. When HSCs were analysed for expression of Mac1 at several time points after transplantation (3, 4, 8 and 16 weeks), we observed an increase in the proportion of Mac1+ cells. To investigate if functional HSC properties are linked to the observed changes in Mac1 expression, we performed secondary transplantations of Mac1+ and Mac1- HSCs. At all times during regeneration, we found robust reconstituting activity in the Mac1+ HSCs. Remarkably, we observed that the reconstituting activity is higher in Mac1+ compared to Mac1- HSCs during the early phase of regeneration. In the late phase of regeneration, however, no difference in reconstitution could be seen. Taken together, our findings suggest that Mac1 expression is associated with functional HSC activity during early regeneration and we are currently investigating in more detail qualitative, as well as quantitative aspects of HSCs in relation to Mac1 expression.
3119 - PDGFRB SIGNALING IS REQUIRED TO GENERATE
AORTIC HAEMATOPOIETIC CELLS IN VIVO Diana Sa da Bandeira1, Zaniah Gonz alez1, Telma Ventura1, 2 Harmen van de Werken , Wilfred van IJcken2, Christer Betsholtz3, Mihaela Crisan1 1 University of Edinburgh, Edinburgh, United Kingdom; 2Erasmus Medical Center, Rotterdam, Netherlands; 3Uppsala University and Karolinska Institute, Uppsala, Sweden
The first hematopoietic stem cells (HSCs) are generated in the aorta-gonads-mesonephros (AGM) region of the mid-gestation mouse embryo. Signals from the microenvironment are required for HSC generation. However, the signals and identity of cells releasing them in vivo remain unknown. The AGM microenvironment includes perivascular cells. Pericytes (PCs) support HSC maintenance in the adult bone marrow, but their involvement in HSC birth has not been studied. PCs are recruited to the developing blood vessel wall via PDGFRb signaling which was recently shown to mediate HSC specification in zebrafish. We hypothesize that PDGFRb signaling is required to generate the first HSCs in vivo. To answer this question, we used the PDGFRb knock-out mice which lack PCs and die perinatally. Results from our hematopoietic progenitor assays (CFU-Cs) and transplantations show that the germline deletion of PDGFRb affects both hematopoietic progenitor numbers and HSC activity in the E11 AGM. HSPCs in other hematopoietic organs are not affected at this stage. Whether PDGFRb+ cells are HSPC precursors or the effect seen is only due to a defect in the niche is under investigation. Characterization of E11 wildtype embryos by immunohistochemistry and RNA sequencing show that the dorsal aorta is surrounded by three phenotypically and transcriptionally distinct perivascular cell layers that include PCs (PDGFRb+NG2+) and PDGFRb+NG2- adjacent cells. PCs are highly enriched in HSC niche genes described in the adult microenvironment, suggesting a putative role in AGM hematopoiesis. In conclusion, our results define PDGFRb signalling as key component of the HSC generating niche in the mouse embryo in vivo that could be tested in vitro to derive HSCs from hematopoietic and non-hematopoietic cell sources for therapy.
3120 - LINEAGE-SPECIFIC RUNX2 SUPER-ENHANCER
ACTIVATES MYC VIA TRANSLOCATION (6;8) AND PROMOTES THE DEVELOPMENT OF BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM Goro Sashida1, Motomi Osato2 1 Kumamoto University, Kumamoto, Japan; 2Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive subtype of acute leukemia, the cell of origin of which is considered to be precursors of plasmacytoid dendritic cells (pDCs). BPDCN cells show high frequencies of mutations in TET2 and p53; however, the molecular mechanisms underlying the pathogenesis of BPDCN have not yet been elucidated. Since translocation (6;8)(p21;q24) is a recurrent anomaly for BPDCN, we demonstrate that a pDC-specific super-enhancer of RUNX2 is associated with the MYC promoter due to t(6;8). RUNX2 ensures the expression of pDC-signature genes in leukemic cells, but also confers survival and proliferative properties in BPDCN cells. Furthermore, the pDC-specific RUNX2 super-enhancer is hijacked to activate MYC in addition to RUNX2 expression, thereby promoting the proliferation of BPDCN, which is reversed by the deletion of super-enhancer of RUNX2 or the inhibition of BRD4 function. We also demonstrate that the transduction of MYC and RUNX2 is sufficient to initiate the transformation of BPDCN in mice lacking Tet2 and Tp53. Since BPDCN cells show lower expression levels of pDCs-signature genes than mature pDCs in patients and mice, we find that macrophage-dendritic cell progenitors, but not mature pDCs, are at least one of the cells of origin of BPDCN by examining the in vivo tumor initiating capacity of those cells. This study provides a model that accurately recapitulates the aggressive human disease and gives an insight into the molecular mechanisms underlying the pathogenesis of BPDCN.
3121 - EFFICIENT AND NON-TOXIC MRNA DELIVERY
AND GENE EDITING IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS USING NANOSTRAWS Ludwig Schmiderer1, Agatheeswaran Subramaniam2, 2 Kristijonas Zemaitis , Alexandra B€ ackstr€ om2, David Yudovich2, Svetlana Soboleva2, Roman Galeev2, Christelle Prinz3, Martin Hjort3 , Jonas Larsson2 1 Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden, Lund, Sweden; 2Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund Unversity, Lund, Sweden; 3Division of Solid State Physics, NanoLund, Lund University, Lund, Sweden Cas9-mediated gene editing of hematopoietic stem cells (HSCs) has great potential for functional studies and gene therapies. Current gene delivery methods, such as viral transduction or electroporation face challenges in clinically relevant parameters, such as efficiency, cell function and viability. We show that nanostraws can be used as an alternative method to deliver small molecules and mRNA to human hematopoietic stem and progenitor cells (HSPCs) without affecting cell viability or colony forming potential. Nanostraws are hollow alumina nanotubes on a polymer membrane, that pierce and connect cultured cells in their native medium with a liquid compartment that contains the cargo of interest. The cargo is delivered into the cells by diffusion or under the influence of a weak electric field. Nanostraws have previously been used to deliver small molecules, mRNA, and proteins to adherent cell lines with high efficiency and low toxicity. We demonstrate that they can also be used to deliver cargo to non-adherent, primary human HSPCs by centrifuging them onto the nanostraws to facilitate close contact and cell-membrane penetration. By applying a pulsed electric field, we delivered propidium iodide and GFP mRNA to more than 75% of treated cells. Treatment with our method did not affect cell viability, proliferative capacity, and colony forming potential, while cells that were treated with conventional electroporation were strongly impaired in these parameters. Further, we used nanostraws to deliver Cas9 mRNA to CD34+ cells and demonstrate successful editing of the CD45 gene. This approach can thus be used to facilitate CRISPR/Cas9 gene editing in primary human HSPCs. Our results highlight that nanostraw mediated cargo delivery is a non-toxic alternative to established methods, with distinct advantages that can be relevant for functional studies and clinical applications.
Poster Presentations / Experimental Hematology 2019;76 (Suppl):S52−S96
3118 - MAC1 AS A POTENTIAL MARKER FOR
FUNCTIONAL HEMATOPOIETIC STEM CELLS DURING EARLY REGENERATION AFTER TRANSPLANTATION Anna Rydstrom, Jonas Larsson Lund University, Lund, Sweden
Mouse hematopoietic stem cells (HSCs) can be isolated to high purity using Lineage-, Sca1+, cKit+, CD48-, CD150+ (LSK SLAM) as markers. Cycling HSCs in the fetal liver have been shown to express the CD11b/CD18 Macrophage-1 antigen (Mac1), a marker expressed mostly on mature cells in the adult bone marrow (Weissman et al., 1995). Furthermore, Mac1 is upregulated in activated adult HSCs after treatment with the cytotoxic drug 5-fluorouracil. For this reason, Mac1 should be omitted from the lineage staining when analyzing fetal liver and 5-FU treated mice. Here we asked whether Mac1 expression is altered also during reconstitution after transplantation into irradiated recipients, and if it can be used to further define the functional HSC compartment in a state of active regeneration. To address this question, LSK cells were transplanted into lethally irradiated (900cGy) primary recipient mice. When HSCs were analysed for expression of Mac1 at several time points after transplantation (3, 4, 8 and 16 weeks), we observed an increase in the proportion of Mac1+ cells. To investigate if functional HSC properties are linked to the observed changes in Mac1 expression, we performed secondary transplantations of Mac1+ and Mac1- HSCs. At all times during regeneration, we found robust reconstituting activity in the Mac1+ HSCs. Remarkably, we observed that the reconstituting activity is higher in Mac1+ compared to Mac1- HSCs during the early phase of regeneration. In the late phase of regeneration, however, no difference in reconstitution could be seen. Taken together, our findings suggest that Mac1 expression is associated with functional HSC activity during early regeneration and we are currently investigating in more detail qualitative, as well as quantitative aspects of HSCs in relation to Mac1 expression.
3119 - PDGFRB SIGNALING IS REQUIRED TO GENERATE
AORTIC HAEMATOPOIETIC CELLS IN VIVO Diana Sa da Bandeira1, Zaniah Gonz alez1, Telma Ventura1, 2 Harmen van de Werken , Wilfred van IJcken2, Christer Betsholtz3, Mihaela Crisan1 1 University of Edinburgh, Edinburgh, United Kingdom; 2Erasmus Medical Center, Rotterdam, Netherlands; 3Uppsala University and Karolinska Institute, Uppsala, Sweden
The first hematopoietic stem cells (HSCs) are generated in the aorta-gonads-mesonephros (AGM) region of the mid-gestation mouse embryo. Signals from the microenvironment are required for HSC generation. However, the signals and identity of cells releasing them in vivo remain unknown. The AGM microenvironment includes perivascular cells. Pericytes (PCs) support HSC maintenance in the adult bone marrow, but their involvement in HSC birth has not been studied. PCs are recruited to the developing blood vessel wall via PDGFRb signaling which was recently shown to mediate HSC specification in zebrafish. We hypothesize that PDGFRb signaling is required to generate the first HSCs in vivo. To answer this question, we used the PDGFRb knock-out mice which lack PCs and die perinatally. Results from our hematopoietic progenitor assays (CFU-Cs) and transplantations show that the germline deletion of PDGFRb affects both hematopoietic progenitor numbers and HSC activity in the E11 AGM. HSPCs in other hematopoietic organs are not affected at this stage. Whether PDGFRb+ cells are HSPC precursors or the effect seen is only due to a defect in the niche is under investigation. Characterization of E11 wildtype embryos by immunohistochemistry and RNA sequencing show that the dorsal aorta is surrounded by three phenotypically and transcriptionally distinct perivascular cell layers that include PCs (PDGFRb+NG2+) and PDGFRb+NG2- adjacent cells. PCs are highly enriched in HSC niche genes described in the adult microenvironment, suggesting a putative role in AGM hematopoiesis. In conclusion, our results define PDGFRb signalling as key component of the HSC generating niche in the mouse embryo in vivo that could be tested in vitro to derive HSCs from hematopoietic and non-hematopoietic cell sources for therapy.
3120 - LINEAGE-SPECIFIC RUNX2 SUPER-ENHANCER
ACTIVATES MYC VIA TRANSLOCATION (6;8) AND PROMOTES THE DEVELOPMENT OF BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM Goro Sashida1, Motomi Osato2 1 Kumamoto University, Kumamoto, Japan; 2Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive subtype of acute leukemia, the cell of origin of which is considered to be precursors of plasmacytoid dendritic cells (pDCs). BPDCN cells show high frequencies of mutations in TET2 and p53; however, the molecular mechanisms underlying the pathogenesis of BPDCN have not yet been elucidated. Since translocation (6;8)(p21;q24) is a recurrent anomaly for BPDCN, we demonstrate that a pDC-specific super-enhancer of RUNX2 is associated with the MYC promoter due to t(6;8). RUNX2 ensures the expression of pDC-signature genes in leukemic cells, but also confers survival and proliferative properties in BPDCN cells. Furthermore, the pDC-specific RUNX2 super-enhancer is hijacked to activate MYC in addition to RUNX2 expression, thereby promoting the proliferation of BPDCN, which is reversed by the deletion of super-enhancer of RUNX2 or the inhibition of BRD4 function. We also demonstrate that the transduction of MYC and RUNX2 is sufficient to initiate the transformation of BPDCN in mice lacking Tet2 and Tp53. Since BPDCN cells show lower expression levels of pDCs-signature genes than mature pDCs in patients and mice, we find that macrophage-dendritic cell progenitors, but not mature pDCs, are at least one of the cells of origin of BPDCN by examining the in vivo tumor initiating capacity of those cells. This study provides a model that accurately recapitulates the aggressive human disease and gives an insight into the molecular mechanisms underlying the pathogenesis of BPDCN.
3121 - EFFICIENT AND NON-TOXIC MRNA DELIVERY
AND GENE EDITING IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS USING NANOSTRAWS Ludwig Schmiderer1, Agatheeswaran Subramaniam2, 2 Kristijonas Zemaitis , Alexandra B€ ackstr€ om2, David Yudovich2, Svetlana Soboleva2, Roman Galeev2, Christelle Prinz3, Martin Hjort3 , Jonas Larsson2 1 Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden, Lund, Sweden; 2Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund Unversity, Lund, Sweden; 3Division of Solid State Physics, NanoLund, Lund University, Lund, Sweden Cas9-mediated gene editing of hematopoietic stem cells (HSCs) has great potential for functional studies and gene therapies. Current gene delivery methods, such as viral transduction or electroporation face challenges in clinically relevant parameters, such as efficiency, cell function and viability. We show that nanostraws can be used as an alternative method to deliver small molecules and mRNA to human hematopoietic stem and progenitor cells (HSPCs) without affecting cell viability or colony forming potential. Nanostraws are hollow alumina nanotubes on a polymer membrane, that pierce and connect cultured cells in their native medium with a liquid compartment that contains the cargo of interest. The cargo is delivered into the cells by diffusion or under the influence of a weak electric field. Nanostraws have previously been used to deliver small molecules, mRNA, and proteins to adherent cell lines with high efficiency and low toxicity. We demonstrate that they can also be used to deliver cargo to non-adherent, primary human HSPCs by centrifuging them onto the nanostraws to facilitate close contact and cell-membrane penetration. By applying a pulsed electric field, we delivered propidium iodide and GFP mRNA to more than 75% of treated cells. Treatment with our method did not affect cell viability, proliferative capacity, and colony forming potential, while cells that were treated with conventional electroporation were strongly impaired in these parameters. Further, we used nanostraws to deliver Cas9 mRNA to CD34+ cells and demonstrate successful editing of the CD45 gene. This approach can thus be used to facilitate CRISPR/Cas9 gene editing in primary human HSPCs. Our results highlight that nanostraw mediated cargo delivery is a non-toxic alternative to established methods, with distinct advantages that can be relevant for functional studies and clinical applications.